Transfer RNA is an integral part of the cell's protein synthesizing system. The biochemical reactions in which tRNA is involved are well documented. Nucleotide sequence analysis, proton NMR spectroscopy, and X-ray diffraction have been used to ellucidate the structure of tRNA. However, the nature of the physical interaction of tRNA with other molecules involved in protein synthesis is essentially unknown. Carbon-13 NMR spectroscopy can be used to study structural and functional interactions of tRNA with other molecules and spectra would be more resolved than the corresponding proton NMR spectra. However, the natural abundance of carbon-13 is very low (1.1 atom %). We propose to specifically enrich tRNA in carbon-13 and then use the (C13)-tRNA for structural and functional studies. tRNA will be enriched in vivo in carbon-13 methyl groups by growing a methionine auxotroph of Escherichia coli in medium with methionine-(methyl(C13)). Specific species of (C13)-tRNA will be purified. In addition specific species of previously unlabeled tRNA will be methylated in vitro using S-adenosyl-L-methionine-(methyl(C13)) and mammalian tRNA-methyltransferases. Labeling the tRNA with (C13)-methyl groups is advantageous because such groups occupy specific, known sites in the tRNA nucleotide sequence, are natural to the molecule and create NMR spectral signals at regions of the spectrum free from ribose and major base interference. Other methods of carbon-13 enrichment of tRNA will be explored utilizing biological systems for the enrichment. Carbon-13 NMR spectroscopy of unfractionated (C13)-enriched tRNA, specific species of (C13)-enriched tRNA and species of tRNA methylated in vitro with (C13)-methyl groups will be used to study the interaction of other nucleic acids and proteins with tRNA at the sites of (C13)-methyl groups.